PB2078: IDENTIFY CHROMOSOME TRANSLOCATION OF CIRCULATING PLASMA CELLS BY CEPHEID GENEXPERT AUTOMATED RT-QPCR WITHOUT FLOW CYTOMETRY AND FISH STAINING
Authors: Tengyao Song, Krupa Shridhar, Mona Liu, Huilin Wei
Journal: HemaSphere (2023)

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: Multiple myeloma (MM) is a malignant B cell neoplasm characterized by uncontrolled proliferation of clonal bone marrow plasma cells (myeloma cells) resulting in bone lesions, anemia, and renal impairment. Therefore, bone marrow aspiration or biopsy remains the widely accepted method to confirm the diagnosis of MM. The classification of MM is determined by cytogenetic analysis of myeloma cells from bone marrow followed by fluorescence in-situ hybridization (FISH) staining. However, the invasive and painful bone marrow biopsy limits the disease burden monitoring which requires repeat sampling. Interestingly, it has been demonstrated that a minimally invasive biomarker from peripheral blood, circulating myeloma cells (CMCs), can reflect tumor burden and predict MM’s clinical outcome. The most common method to identify CMCs includes multi-color flow cytometry which requires complex and expensive equipment that makes it unfeasible for developing countries with technical and financial challenges. FISH staining is a standard method to identify chromosome abnormality with the known limitation of being time-consuming and low throughput. It has been reported that IGH-related translocation is characterized by the unique overexpression of the target gene. Therefore, using multiplex qRT-PCR to measure the relative expression of the target gene from myeloma cells provides a rapid assay to detect chromosome translocation. Aims: Introducing an assay that combines rapid isolation of circulating myeloma cells (CMCs) from peripheral blood and a multiplex qRT-PCR to detect common chromosome translocation in CMCs. Methods: We introduce a two-step CMCs isolation process: 1. utilizing an antibody cocktail to deplete the unwanted cells from peripheral blood: RBCs, macrophages, T cells, B cells, and platelet. 2. enrich CD138+ myeloma cells with CD138 monoclonal antibody-coupled magnet beads. The IGH-related translocation target gene expression is measured and normalized with the housekeeping gene by Cepheid GeneXpet System: MMSET and FGFR3 for t(4;14), CCND1 for t(11;14), MAF and ITGB7 for t(14;16), MAFB and ITGB7 for t(14;20), CCND3 for t(6;14), and ABL for housekeeping gene. The types of translocations can be detected by the unique target gene expression profile from each translocation. Results: we present a novel method that identifies the chromosome translocation of CMCs from peripheral blood without flow cytometry or FISH staining: a. our method rapidly isolated CMCs by antibody-based positive and negative cell selection which achieves the LOD of 0.005%. b. We utilize our GeneXpert Instrument System, a single-use cartridge product with pre-loaded sample processing reagents that automates the RT-qPCR to identify CMCs cytogenetics, with turn-around time to results of less than 4 hours, including hands-on time. c. the number of CMCs can be quantified to mirror the course of the disease and response to therapy. Summary/Conclusion: We have successfully developed an assay prototype with the following highlights: a. Without flow cytometry, the assay achieves the LOD of 0.005% (300 myeloma cells/ml of peripheral blood) which is comparable to the threshold for CMCs detection by flow cytometry. b. Without FISH staining, the assay detects chromosome translocation by assessing the relative target gene expression profile in the fully automated GeneXpert System leading to a faster turn-around time. Keywords: Quantitative RT-PCR, Chromosomal translocation, Multiple myeloma, Diagnosis